内皮细胞内质网应激-自噬激活在肉毒毒素A预处理游离皮瓣拮抗再灌注损伤中作用机制研究

Autophagy is regarded as an important mechanism which could provide the stability and surviving of the endogenous environments under the cellular stress.We found the activation of the autophagy of the endothelial cell could fight against the reperfusion injury of the skin flap in previous study；Botox A preconditioning the free skin flap could activate the protective autophagy of the endothelial cell. Further investigation revealed that Hypoxia/ reoxygenation endothelial cell could prompt the more expression of GPR78. Bertolotti A suggested that GPR78 plays a critical role which activates the ER stress. We hypothesis that Botox A preconditioned the skin flap with the activation of the ER stress of the endothelial cell, later the protective autophagy rose against the reperfusion injury. We made a plan that we would precondition the ischemia/reperfusion injury model of the skin flap with Botox A, observe the autophagy bodies and apoptotic index, and confirm the activation of the autophagy of the endothelial cell is a key factor during the preconditioning of the skin flap with Botox A on the overall level; precondition the hypoxia/reoxyenation endothelial cell with botox A, observe the changing law of the autophagy at different times, and explore the character of the dose effect and time effect on the cellular level; regulate the message path of ER and observe the protein of different path ,and illustrate the mechanism of ER stress on the autophagy on the molecular level. The study will confirm autophagy plays a critical role during the preconditioning of the skin flap with botox A, to explore more clinical usage of botox A.

自噬是细胞应激情况下维持内环境稳定和存活重要机制。前期发现内皮细胞自噬激活可以拮抗皮瓣缺血再灌注损伤；肉毒毒素A预处理游离皮瓣可以激活内皮细胞自噬保护皮瓣。进一步发现低氧/复氧内皮细胞模型行肉毒素A处理后GPR78表达增加。Bertolotti A 认为GPR78是调控内质网应激启动的关键分子。我们假说肉毒毒素预处理皮瓣后激活内质网应激，然后启动内皮细胞保护性自噬可拮抗皮瓣再灌注损伤。拟肉毒毒素A预处理皮瓣再灌注损伤模型，观察内皮自噬体和凋亡指数等，整体水平明确内皮自噬激活是肉毒毒素A预处理抗皮瓣凋亡关键；低氧/复氧内皮细胞模型予以肉毒毒素A处理，不同时间段观察自噬变化规律，细胞水平探讨自噬变化的时效和量效特点；调控内质网信号通路，监测内质网不同通路蛋白，分子水平阐明内质网应激调控自噬的信号机制。研究将明确内皮自噬激活是对游离皮瓣行肉毒素A预处理保护关键靶点，拓展肉毒毒素A的临床用途。

自噬是细胞应激情况下维持内环境稳定和存活的重要机制，本研究项目从动物模型和细胞实验两个水平探讨肉毒毒素A预处理保护游离皮瓣的机制。在动物实验水平，游离皮瓣发生缺血再灌注损伤后皮瓣坏死面积明显增加，而肉毒毒素A预处理后游离皮瓣的坏死面积及死亡率明显降低、血管数量增加、血管内皮形态趋于规则，但是联合应用自噬抑制剂3-MA后，肉毒毒素A的这种作用被逆转；在细胞实验水平，采用不同浓度的肉毒毒素A进行预处理，结合凋亡率及凋亡相关蛋白的表达结果，发现肉毒毒素A的保护作用具有剂量依赖性；然后将内皮细胞分为4组：对照组、缺氧/复氧组、肉毒毒素A预处理组、肉毒毒素A预处理联合应用自噬抑制剂CQ组，采用Western Blot检测自噬蛋白LC3Ⅱ/Ⅰ和Beclin1的表达，流式细胞仪检测内皮细胞的凋亡率，免疫荧光检测自噬蛋白的表达，透射电子显微镜检测自噬小体的形态结构，发现肉毒毒素A预处理组的凋亡率降低、LC3Ⅱ/Ⅰ和Beclin1的表达增加，LC3的免疫荧光强度增强、自噬小体数量增加。进一步实验发现肉毒毒素A预处理后，内质网应激蛋白GRP78表达增加，内质网应激蛋白的表达也发生改变。本研究项目明确内皮自噬激活是对游离皮瓣行肉毒素A预处理保护关键靶点，拓展肉毒毒素A的临床用途。